PDO is an important chemical material which can be used in printing, dyeing, coating, lubricant, and antifreeze industries as an organic solvent. PDO is widely used as monomer in polyester and polyurethane synthesis. Especially, poly(trimethylene terephthalate) (PTT), which is formed by polymerization of PDO with terephthalic acid, has been shown to exhibit superior properties over the polymers formed between terephthalic acid with either 1,2-propanediol, butanediol, or glycol as monomer. Annually, tens of million tons of poly(ethylene terephthalate) (PET) are consumed globally. The chemical stability and biodegradability of PTT are comparable to that of PET, but PTT is superior to PET in some aspects, such as resistance to pollution, ductility and resilience, and anti-UV properties. Furthermore, PTT fiber has advantages of wear resistance, low moisture absorption and low electrostatic generation, making it a potent competitor of Nylon in carpet industry. Additionally, PTT can also be used in the fields of nonwovens, engineering plastics, costume, domestic decoration, padding materials, and wovens. PTT was once selected as one of the six novel petrochemical products in USA in 1998, and therefore regarded as an up-graded product for PET.
The superior properties and commercial value of PTT were recognized as early as 50 years ago. However, since this raw material PDO is hard to produce, which also means a high production cost of PDO, it has been difficult to produce PDO on an industrial scale. So far, only DuPont and Shell could produce PDO by traditional synthesis method from ethylene oxide or propylene, whereas the PDO produced in this way is mainly used by these two companies to produce PTT. This traditional synthesis method suffers from defects of a plurality of undesired byproducts, unsatisfactory selectivity, severe operation conditions of high temperature and high pressure, and huge investment on installations. Further, the raw materials thereof are non-renewable, ethylene oxide is inflammable and explosive, and one of the intermediate material acrolein is extremely toxic. Contrarily, PDO fermentation production method is of great interest recently due to its selectivity and mild operation conditions.
Biosynthetic production of PDO utilizes micro-organisms in which glycerol can be converted into PDO via dismutation. Among the naturally occurring micro-organisms which can convert glycerol into PDO and are mainly anaerobes or facultative anaerobes, Klebsiella pneumoniae, Clostridium butyricum, and Citrobacter freundii show a relatively higher rate of PDO conversion and are more tolerant to glycerol and the product PDO, and thus are promising micro-organisms for future development and application in this area.
BDO, which is a byproduct of the process of fermentation production of PDO, is also an important chemical material. BDO, a colorless, odorless liquid, can be used as fuel or be used to prepare polymers, printing ink, perfume, antifreeze, fumigant, humectant, softening agent, plasticizer, dynamite, and chiral ionophore for pharmaceuticals. BDO may also be used as a valuable chemical material to produce other chemicals. For example, BDO can be converted into methylethylketone via dehydration, which has a wide application, for example, it can be converted into 1,3-butadiene upon further dehydration. BDO can form Styrene by going through Diels-Alder reaction. A condensation reaction occurred between BDO and methylethylketone followed by a hydrogenation reaction results in the production of octane, which is a high quality aviation fuel. A reaction between BDO and acetic acid produces 2,3-butanediol diacetate, which can be used as an additive to improve the flavor of cream. Nonetheless, BDO is generally not isolated and purified during the process of fermentation production of PDO as its yield is relatively low.
Polyhydroxyalkanoates (PHAs) refer to biopolymers generated from β-hydroxy fatty acid monomers via esterification. At least 125 different types of PHA polyester-forming monomer structures have been found, with new monomers being identified continuously. PHAs synthesized in micro-organisms exhibit certain special properties, including biodegradability, biocompatibility, piezoelectricity and optical activities. PHAs are suitable to use in many types of human tissue and organs, e.g., cardiovascular system, cornea, pancreas, gastric-enteric system, kidney, genitourinary system, musculo skeletal system, nervous system, dental and oral tissues, skin, etc. Commercially available PHA products mainly include PHB, PHBV and PHBHHx. Moreover, PHAs can be rigid or soft or flexible, according to the structures and contents of its monomers. PHAs may have many potential applications, on which both basic research and application development studies have been extensively carried out.
The simplest PHA-forming monomer is β-hydroxypropionic acid, and the polymer generated by polymerization of β-hydroxypropionic acid monomers is Polyhydroxypropionic acid (PHP). It has been found that the intensity of PHP is higher than that of other types of PHAs, and thus it may find potential applications in some areas. The monomer directly utilized in the biosynthesis of PHA is β-hydroxy fatty acyl coenzyme A, a form of monomer containing high-energy chemical bond. PHP can be produced by the conversion of intermediate products of glycerol metabolism in micro-organisms (FIG. 1).
In the 1980s, only two PHAs, i.e., PHB (Chemie Linz A G) and PHBV (Zeneca), a co-polymer of hydroxybutyric acid and hydroxyvaleric acid, achieved an industrial scale production. In 1998, a collaborative group from Laboratory of Microbiology, Tsinghua University and Guangdong hangmen Center for Biotech Development Co., Ltd successfully developed a process of industrially producing PHBHHx, a co-polymer of hydroxybutyric acid and hydroxyhexanoic acid, laying a material foundation for the application of this new material.
There are several problems involved in the process of producing PDO by fermentation. For example, at the late stage of fermentation, cell growth arrest, reduced PDO increase, and lactic acid accumulation will generally occur, and a large amount of accumulated lactic acid will make the extraction of PDO more difficult. In addition, the decrease in activity of glycerol dehydrase and insufficiency of intracellular Coenzyme NADH2 at the late stage of fermentation are also the reasons leading to the reduced PDO increase. It is true that higher concentrations of PDO may be achieved by prolonging the course of fermentation, but this will inevitably increase the cost of production. The above issues are the impediments to industrial scale production of PDO. To solve these problems, one of the current strategies is to obtain genetically modified strains which can improve the level of PDO production by fermentation or achieve combined production of other high value-added products, so as to substantially reduce the cost of PDO production.
Currently, efforts on genetic modification of wild-type strains are mainly focused on the following aspects:
(1) To increase the expression of rate-limiting enzymes (e.g., glycerol dehydrase, PDO oxydoreductase) in the reduction pathway by the means of genetic engineering:
Zeng et al [Sun J B., Heuvel J., Soucaille P., Qu Y., and Zeng A. P. Comparative Genomic Analysis of dha Regulon and Related Genes for Anaerobic Glycerol Metabolism in Bacteria. Biotechnol. Prog. 2003 19:263-272] constructed a plasmid containing genes encoding glycerol dehydrase and PDO oxydoreductase and inserted it into a wild-type strain. It was found that the activities of both enzymes in the strain were largely elevated. However, this genetically engineered strain failed to produce higher concentration of PDO during fermentation. By using a new cloning method, HUANG Ribo et al inserted a glycerol dehydrase gene into an E. coli strain. It was shown that the resultant strain was capable of producing PDO in a concentration of 30-35 g/L, and the yield of PDO vs. glycerol is about 40% [HUANG Ri-bo et al, Clostridium perfringen glycerol dehydrase gene, and its use in 1,3-propylene glycol producing method. Chinese Patent Application No.: 200610019452.X (CN1935991)].
(2) To knockout genes encoding adverse products and block the pathway of by-product metabolism:
ZHANG Yan-ping et at [ZHANG Yan-ping LIU Ming CAO Zhu-an. Construction of K. pneumoniae Recombinants of Aldehyde Dehydrogenase Gene Knockout. China Biotechnology, 2005, 25(12):34-38] obtained two recombinant strains by deleting the Aldehyde dehydrogenase (ALDH) gene of ethanol synthesis pathway in K. pneumoniae M5aL using homologous recombination technique. The results of batch fermentation experiments under anaerobic conditions showed that yields of ethanol of recombinants were decreased by 43%˜53% and yields of PDO were increased by 27%˜42%, comparing with those of the wild-type K. pneumoniae M5aL. But the final concentration of PDO was only 16 g/L. YANG Guang constructed genetically engineered K. pneumoniae M5aL strains respectively lacking acetic acid, ethanol and lactic acid metabolism pathways. Although the rate of glycerol conversion was increased, the final concentration and productivity of PDO were decreased [YANG Guang, Molecular Breeding of Klebsiella pneumoniae for 1,3-propanediol Production. Beijing: China Agricultural University, 2003 [Thesis]].
(3) To construct Coenzyme regeneration system in PDO producing strains:
HUANG Zhi-hua, et al [HUANG Zhi-hua, ZHANG Yan-ping, CAO Zhu-an. Expression and functional analysis of Formate dehydrogenase in Klebsiella pneumoniae. Acta Microbiologica Sinica, 2007, 47 (1): 64-68] isolated a gene from the genome of C. boidinii encoding a formate dehydrogenase which is capable of regenerating reduced Coenzyme I (NADH2). They constructed a recombinant plasmid containing the formate dehydrogenase gene, and for the first time constructed a NADH2 regenerating system in a PDO producing strain K. pneumoniae. The concentration of 1,3-PDO produced in this K. pneumoniae strain transformed with the recombinant plasmid achieved 78.6 g/L, which is 12.5% higher than that of the starting strain YMU2. HUANG Zhi-hua, et al [HUANG Zhi-hua; ZHANG Yan-ping; HUANG Xing; Wang Bao-guang; CAO Zhu-an. Construction of NADH Regeneration System in Klebisella pneumoniae with Aldehyde Dehydrogenase Inactivated. China Biotechnology, 2006, 26 (12): 75-80] transformed the recombinant plasmid containing formate dehydrogenase gene into an Aldehyde dehydrogenase inactivated Klebsiella pneumoniae DA 21HB strain. The concentration of PDO produced in this recombinant strain achieved 75.06 g/L, which is 19.2% higher than that of the starting strain DA 21HB.
(4) To construct genetically engineered E. coli strains which utilize glucose to produce PDO:
DuPont and Genencor have obtained a number of patents in the field of constructing biocatalysts using glucose as substrate [Bulthuis B A, Gatenby A A, Haynie S L, et al. Method for the Production of Glycerol by Recombinant Organisms. U.S. Pat. No. 6,358,716, 2002-05-19. Diaz-Torres M, Dunn-Coleman N S, Chase M W, et al. Method for the Recombinant Production of 1,3-Propanediol. U.S. Pat. No. 6,136,576, 2000-10-24. Emptage M, Haynie S L, Laffend L A, et al. Process for the Biological Production of 1,3-Propanediol with High Titer. U.S. Pat. No. 6,514,733, 2003-08-21.], in which an genetically engineered strain with high yields under aerobic conditions was obtained by using E. coli K12 as starting strain. This strain was tested in fed-batch fermentation experiments and the concentration of 1,3-PD produced in fermentation is 135 g/L. A drawback of this strain is that it is Coenzyme B12 dependent, and thus the production cost is relatively high.
(5) To construct genetically engineered PDO-producing strains from glycerol-producing bacteria:
Cameron et al [Cameron D C, Altaras N E, Hoffman M L et. al. Metabolic Engineering of Propanediol Pathways. Biotechnol. Prog. 1998, 14: 116-125] constructed a Saccharomyces cerevisia strain expressing the genes encoding these two enzymes from Klebsiella pneumoniae. Fermentation was carried out under anaerobic conditions in a culture medium supplemented with Vitamin B12 and containing 5 g/L of glucose as carbon source, but no detectable PDO was found in the broth during 48 hours of fermentation.